Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Volume of Primers

Primers qPCR PCR

  • Please log in to reply
4 replies to this topic

#1 Mad Researcher

Mad Researcher

    Newbie !!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 193 posts
12
Good

Posted 02 May 2015 - 03:10 PM

Please correct me if i am wrong or kindly suggest a way to solve this:

 

I want to calculate the volume of primer (microliter) that i need to use for PCR reaction:

 

x = final conc. of primer X total vol. of reaction/ stock conc. of primer

x = 0.3 * 20/10 = 0.6 microliter of primer

 

 


Cheers,

Mad Researcher

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,083 posts
456
Excellent

Posted 03 May 2015 - 12:58 PM

Looks fine... it's just a rearrangement of C1V1=C2V2.



#3 Mad Researcher

Mad Researcher

    Newbie !!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 193 posts
12
Good

Posted 03 May 2015 - 01:14 PM

Looks fine... it's just a rearrangement of C1V1=C2V2.

 

Thanks bob, I also thought the same.

Its V1= c2v2/c1


Cheers,

Mad Researcher

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,673 posts
282
Excellent

Posted 03 May 2015 - 07:09 PM

It REALLY helps if you use units with your math. Units cancel, just like numbers, and it makes many mysterious things obvious. I would never have written the equation as you did. Rather:

 

x ul = 0.3 uM * 20 ul / 10 uM

 

The uM cancels, and you have x ul = 0.6 ul

 

If your  units fail to cancel in the right way, your equation is wrong.



#5 Leishman001

Leishman001

    member

  • Active Members
  • Pip
  • 17 posts
1
Neutral

Posted 04 May 2015 - 12:58 PM

No comment on your math, I think the other scientists covered it.

 

Next time you order primers, or if you have a master stock that you can dilute, take note of the amount of DNA (usually in nanomoles) that the company delivers to you. Calculate the volume of solvent (water or Tris pH 8) to add to make a 100 pmol per ul stock. I typically add 100 pmol of each primer (forward and reverse) per reaction, so I just add 1 ul of each primer to the reaction mix. Less calculation AND you don't have to pipet less than 1 ul.

 

For example, if the primer company delivers 350 nmol of primer (typical amount), add 3500 ul of buffer to make 0.1 nmol/ul or 100 pmol/ul.







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.